Defects in the Mre11-Rad50-Nbs1 (MRN) complex results in human disease including cancer, and severe DNA damage phenotypes in yeast. MRN, acting with CtIP, is essential for the repair of double- stranded DNA breaks (DSBs) by homologous recombinational repair (HRR), as well as acting in meiosis, antibody hypermutation, telomere maintenance, rescue of stalled replication forks, and DNA damage signaling through ATM kinase. Yet, the mechanistic basis for diverse MRN functions is poorly understood. We propose three Specific Aims to understand MRN, CtIP and ATM structural biochemistry, activities, conformations and interactions relevant for DSB repair and signaling. We will couple advanced biophysical technologies, including atomic-resolution crystal structures and small- angle X-ray scattering in solution, with mutagenesis, biochemistry and yeast genetic analyses. Our integrated approaches will test hypotheses that dynamic MRN conformations and macromolecular interfaces control biological responses at DSBs. In particular, our results will significantly advance knowledge of 1) Mre11 DNA-binding and nuclease mechanisms and their importance for DNA repair pathway choice and progression. 2) How Rad50 binds to DNA and uses its ATPase activity to both handoff DNA to Mre11 and allosterically regulate Mre11 nuclease activities. 3) A Rad50 patient mutation that will advance our understanding of therapeutically targetable Rad50 protein features. 4) Catalytic and non-catalytic roles of CtIP. 5) How MRN recruits and activates ATM at DSBs. Our latest Mre11 inhibitor results and work from others in the field suggest that MRN roles in DSB repair and signaling are viable targets for the development of advanced adjunct cancer therapies, which work by synthetic lethality with current radiation and chemotherapies along with weaknesses in other DNA repair or signaling pathways arising from either inhibitors or cancer-specific genetic defects. Thus, our integrated results will provide a molecular framework for understanding cancer etiologies from DNA repair defects and for the design of advanced cancer therapies targeted against specific MRN activities. Collectively, project results will connect MRN, CtIP and ATM to cellular outcomes and human disease-states by defining interactions, conformations and mechanisms critical for genetic integrity, cancer therapy resistance, and future cancer treatments.